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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 10 4832-4839
Copyright © 2003 by The Endocrine Society

Enhanced Repression by HESX1 as a Cause of Hypopituitarism and Septooptic Dysplasia

Ronald N. Cohen, Laurie E. Cohen, Diego Botero, Christine Yu, Angela Sagar, Magdalena Jurkiewicz and Sally Radovick

Section of Endocrinology (R.N.C., C.Y., A.S., M.J.), Department of Medicine, University of Chicago, Chicago, Illinois 60637; Division of Endocrinology (L.E.C., D.B.), Department of Medicine, Children’s Hospital, Boston, Massachusetts 02115; and Section of Pediatric Endocrinology (C.Y., S.R.), Department of Pediatrics, University of Chicago, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Ronald Cohen, M.D., Section of Endocrinology, Department of Medicine, 5841 South Maryland Avenue, MC 1027, Chicago, Illinois 60637. E-mail: roncohen{at}medicine.bsd.uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HESX1 is a paired-like homeodomain transcription factor that functions as a repressor of PROP1-mediated gene stimulation. Mutations in HESX1 have been implicated in cases of septooptic dysplasia and congenital hypopituitarism. All mutations in HESX1 identified to date have resulted in impaired DNA binding and defective HESX1 action. We have identified a novel HESX1 mutation in genomic nucleotide position 1684 (g.1684delG), which results in a mutant protein with increased DNA binding. In turn, this mutation causes increased repression of PROP1-dependent gene activity. These data suggest that enhancement of transcriptional repression during pituitary organogenesis is a novel mechanism for the development of congenital pituitary disorders.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DEVELOPMENT OF the anterior pituitary gland proceeds via a tightly regulated program characterized by the graded expression of transcription factors and signaling molecules (1). Such factors are expressed in distinct spatial and temporal patterns during organogenesis. This ultimately leads to the formation of the mature anterior pituitary, with its distinct hormone-producing cell types: thyrotrophs, gonadotrophs, lactotrophs, somatotrophs, and corticotrophs. It has become clear that mutations in a number of developmentally important pituitary-specific transcription factors lead to hypopituitarism (2). For example, POU1F1 (also known as Pit-1), a pituitary-specific transcription factor, is important for the development of thyrotrophs, somatotrophs, and lactotrophs (3), and POU1F1 mutations have been identified in patients with variable degrees of deficiencies of these cell types (4, 5, 6, 7). Patients with PROP1 mutations have similar phenotypes and may have loss of gonadotropin secretion with age and progressive ACTH deficiency (2, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17), although not all patients with PROP1 mutations develop ACTH deficiency.

HESX1 (also called Rpx) is a paired-like homeodomain transcription factor with repressing domains located in the N terminus and homeodomain (18, 19). Expression of Hesx1 in the developing mouse initially occurs during gastrulation; it later becomes restricted to Rathke’s pouch, the precursor of the pituitary gland (18). Expression decreases by embryonic d (E)13 and becomes undetectable by E14–E15.5 (20). Down-regulation of HESX1 appears to correlate with the production of differentiated pituitary cell types. Although the exact function of HESX1 is not clear, its repressing function appears to be vital. The N-terminal repressing domain of HESX1 binds TLE1, a mammalian homolog of Groucho (19); the homeodomain also interacts with the nuclear corepressor (NCoR) (21). NCoR was initially identified for its role in nuclear hormone receptor action (22) but has been found to interact with a variety of other transcriptional repressors.

HESX1 is closely related to another pituitary-specific paired-like homeodomain factor, PROP1 (20), which functions as a potent activator. Both transcription factors bind to the same DNA response elements, for example, the PRDQ9 sequence, which is an idealized PROP1 and HESX1 binding site (20). Dasen et al. (19) have proposed that HESX1 and PROP1 function as opposing transcription factors and that the carefully regulated temporal sequence of their expression is vital for normal pituitary development. For example, PROP1-dependent regulation of pituitary development occurs during the attenuation of HESX1 expression. Furthermore, premature expression of PROP1 leads to pituitary abnormalities, as does prolonged expression of HESX1 and TLE1, suggesting that the switch from HESX1 to PROP1 must be carefully timed for normal development to occur (19).

Mutations in HESX1 have been implicated in cases of septooptic dysplasia (SOD) and hypopituitarism in humans (23, 24, 25). SOD is characterized by optic nerve hypoplasia, pituitary gland dysfunction, and midline abnormalities involving the corpus callosum and septum pellucidum (2, 24). Targeted disruption of HESX1 in mice causes a similar phenotype (19, 24). All previous cases of SOD shown to be caused by mutations in HESX1 are the result of mutations in the homeodomain of the protein, leading to a transcription factor that does not bind DNA and is thus inactive (23, 24, 25). In particular, the initial cases that were reported involved two siblings with homozygous R160C mutations; these patients had agenesis of the corpus callosum and panhypopituitarism (24). Interestingly, more recently described heterozygous HESX1 mutations have been associated with variable, and more mild, pituitary phenotypes, including isolated GH deficiency (23).

We have identified a patient with SOD with a novel heterozygous HESX1 mutation. This mutation is distinct from previously reported mutations. It results in the improved ability of the mutant HESX1 protein to block PROP1-dependent gene transcription via enhanced DNA binding. This is the first example of a mutation leading to hypopituitarism as a result of enhanced repression. These data reinforce the notion that the carefully timed expression of repressors is vital during development. Specifically, repression plays a vital role in pituitary organogenesis, and disruption of the normal pattern of repression in the developing gland leads to the development of pituitary hypofunction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction and mutational analysis

This study was approved by the Institutional Review Boards at Children’s Hospital (Boston, MA) and The University of Chicago (Chicago, IL), and informed consent was obtained. Genomic DNA was isolated from the patient with SOD. Each HESX1 exon was amplified by PCR, subcloned, and sequenced. In addition, the full-length genomic HESX1 sequence was amplified from wild-type genomic DNA and inserted into the expression vector pSG5. This vector was transfected into human 293T cells, mRNA was isolated, and a HESX1 cDNA clone was obtained by RT-PCR and cloned again into the expression vector pSG5, modified to have an N-terminal FLAG tag. The mutation was inserted by PCR. Wild-type and mutant HESX1 were also placed in-frame downstream of the Gal4 DNA-binding domain in the expression vector pECE. Wild-type PROP1 genomic DNA and cDNA clones were obtained using a similar approach. Three copies of a PRDQ9 sequence were constructed with oligonucleotides; sense and antisense strands were annealed and placed into the luciferase-containing pGL2-promoter vector (Promega, Madison, WI).

Transient transfection and cell culture

Transient transfections were performed in 293T cells; culture conditions were as previously described (26). Transient transfections were performed in six-well plates using the calcium phosphate technique. For functional studies, the pSG5-PROP1 genomic construct was used. A total of 330 ng of a PRDQ9-luciferase vector was cotransfected with 60 ng pSG5-genomic PROP1 with or without 0–60 ng of pSG5-wild-type or mutant HESX1. All transfections were balanced with the appropriate amount of empty vector pSG5. In addition, 7 ng of a Renilla luciferase construct (Promega) were cotransfected to control for transfection efficiency. For experiments using Gal4 constructs, 660 ng of an upstream activating sequence (UAS)-Luciferase reporter was cotransfected with 150 ng Gal4-HESX1 construct (wild-type or mutant) or empty Gal4, along with 660 ng of pSG5 or pSG5-genomic PROP1 (where indicated). For the Gal4 experiments, 30 ng of a cytomegalovirus (CMV) ß-galactosidase vector was cotransfected to each well to control for transfection efficiency. Sixteen to 24 h after transfection, the media was changed to fresh media; about 48 h after transfection, the cells were lysed and assayed for luciferase activity. Luciferase measurements were divided by Renilla luciferase (or ß-galactosidase) activity to control for transfection efficiency. Data are expressed as relative luciferase activity (±SEM). Experiments were performed at least three times in triplicate.

EMSA

EMSA was performed as previously described (26). Oligonucleotides containing a PRDQ9 element were annealed and 32P-labeled with polynucleotide kinase. The sense strand was 5'-agcttgagactaattgaattagcctgtac-3'. HESX1, mutant HESX1, and PROP1 cDNA constructs in pSG5 were in vitro translated using a coupled transcription/translation system in reticulocyte lysate (Promega) with T7 polymerase. For each EMSA, in vitro translated proteins were mixed with radiolabeled probe for 20 min at room temperature, then run on a 5% nondenaturing gel at 4C, and analyzed by autoradiography. Proteins were also in vitro translated with 35S methionine and analyzed by SDS-PAGE.

Western blot analysis

Either 2 or 10 µg of pSG5-HESX1 or pSG5-mutant HESX1 was transfected into 293T cells. Twenty-four hours after transfection, cells were washed in PBS and changed to fresh media. Forty-eight hours after transfection, proteins from whole cell extracts were isolated, run on SDS-PAGE, transferred to nitrocellulose, and blotted using an anti-FLAG antibody (Sigma, St. Louis, MO). The results were visualized by ECL+ (Amersham Pharmacia Biotech, Piscataway, NJ).

Northern blot analysis

The indicated amount of wild-type or mutant HESX1 construct in pSG5 was transfected into 293T cells. Fifteen to 18 h later, cells were washed in PBS, and new media was added. Forty to 44 h after transfection, mRNA was isolated (Invitrogen, San Diego, CA). Equivalent amounts of mRNA were loaded on a gel, transferred, and blotted with a probe representing the N-terminal portion of HESX1 (in common to both the wild-type and mutant mRNA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Patient phenotype and characterization

The patient was diagnosed with SOD at 6 months of age, when he presented with blindness and nystagmus. Magnetic resonance imaging revealed an absent corpus callosum, thin optic nerves, small anterior pituitary gland, and absence of the posterior bright spot. He first presented to the Endocrine Program at Children’s Hospital Boston at the age of 3 yr and 6 months. Family history was negative for endocrine disorders, dysmorphic features, or metabolic conditions. There was no consanguinity between the parents. On physical examination, he had bilateral wandering nystagmus. There were no midline facial defects, and he had normal prepubertal genitalia. He had no evidence of GH inadequacy, because height was 98.2 cm (SD score, -0.14); weight, 16.8 kg (SD score, +0.68); IGF-I, 50.0 ng/ml (50 µg/liter; normal, 17–248 ng/ml); IGF binding protein-3, 1.8 mg/liter (normal, 1.4–3.0 mg/liter); and bone age, not delayed at 4 yr. Thyroid function was normal, with T4, 7.4 µg/dl (95.5 nmol/liter; normal, 6.9–13.0 µg/dl); thyroid binding globulin index (also known as the thyroid hormone binding ratio, a normalized version of the T3 Resin Uptake), 0.99 (normal, 0.89–1.13); and TSH, 1.84 µU/ml (1.84 mU/liter; normal, 0.3–6.2 µU/ml). He had no complaints of polydipsia or polyuria, and his serum osmolarity of 279 mOsm/kg (279 mmol/kg) and urine osmolarity of 830 mOsm/kg (830 mmol/kg) under basal conditions revealed the ability to concentrate his urine and the lack of diabetes insipidus. He previously had a 250-µg ACTH-(1–24) stimulation test performed, which was normal with a baseline cortisol level of 5.0 µg/dl (140 nmol/liter) rising to 21.7 µg/dl (607.6 nmol/liter) 60 min after cosyntropin.

At the age of 4 yr and 9 months, he was noted to have a decreasing growth velocity with a low IGF-I level of 22 ng/ml. Prolactin (PRL) and TSH levels were normal (including responses to TRH stimulation), and the cortisol response to insulin-induced hypoglycemia was intact with a baseline level of 8.8 µg/dl (246.4 nmol/liter) that rose to 20.8 µg/dl (582.4 nmol/liter) (Fig. 1AGo). However, the peak GH response to insulin-induced hypoglycemia was inadequate at 4.2 ng/ml (4.2 µg/lilter) (Fig. 1AGo). His growth rate fell to 2.2 cm/yr. He was prescribed recombinant human GH at a dose of 0.18 mg/kg·wk for GH deficiency with excellent catch-up growth. Most recently at age 6 yr, thyroid hormone level was borderline, with TSH, 5.13 µU/ml (5.13 mU/liter; normal, 0.3–6.2 µU/ml); T4, 6.4 µg/dl (82.6 nmol/liter; normal, 6.0–12.3 µg/dl); and thyroid binding globulin index, 0.91 (normal, 0.84–1.08) for a calculated T4 index of 5.8.



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  		FIG. 1. Clinical data and amino acid sequence comparison of wild-type and mutant HESX1. A, Clinical data from a TRH stimulation test and an insulin tolerance test on the patient. The GH response to insulin is inadequate. To convert TSH values to milliunits per liter, no change is necessary; to convert PRL to picomoles per liter, multiply by 44.4; to convert GH to micrograms per liter, no change is necessary; to convert cortisol to nanomoles per liter, multiply by 28. B, A schematic diagram of the human HESX1 protein is shown, along with the amino acids that are changed as a result of the nucleotide deletion in mutant HESX1. The amino acid sequences of the C-terminal portion of wild-type and mutant HESX1 are shown. The amino acids in the homeodomain are as indicated in Dattani et al. (24 ). The amino acid sequence of mutant HESX1 was deduced from the change in the DNA coding sequence. The frameshift mutation causes an alteration in the C-terminal portion of the protein.

 
Sequencing analysis revealed a heterozygous deletion of guanine in what would be codon 175 of wild-type HESX1, resulting in a frameshift mutation and novel C-terminal portion of the protein (Fig. 1BGo). This base corresponds to nucleotide 1684 (g.1684delG) of the HESX1 gene (GenBank accession no. AF059734), which is located in exon 4. Interestingly, the anticipated length of the mutant HESX1 is one amino acid longer than the wild-type protein. No other mutations were identified in other exons or in intron-exon borders. The novel C-terminal amino acids do not create known protein motifs with specific functions. The amino acid differences lie C-terminal to the region identified as the homeodomain (24). Sequencing analysis of the mother’s HESX1 sequence did not reveal a mutation; the father is not available for analysis. The mother’s height is 5 feet 1 inch; the father’s approximate height is 5 feet 9 inches. There are no known vision problems in either parent. There are no siblings.

Mutant HESX1 is more potent in blocking PROP1-mediated gene transcription

To develop a functional assay for the effects of wild-type and mutant HESX1, a reporter was constructed with three copies of the PRDQ9 sequence upstream of luciferase. The PRDQ9 sequence was initially identified as an idealized binding site for HESX1 and PROP1; similar sequences are present in the POU1F1 promoter (20). Transfection of pSG5-HESX1 alone with PRDQ9-Luc has minimal effects (data not shown), as previously reported (20). However, cotransfection of pSG5-HESX1 with pSG5-PROP1 results in an inhibition of PROP1-dependent luciferase activity. Transfection of pSG5-PROP1 with PRDQ9-Luc results in a 6- to 7-fold stimulation of luciferase activity (Fig. 2AGo), and cotransfection of pSG5-HESX1 and pSG5-PROP1 decreases PROP1-dependent luciferase activity, with increasing amounts of pSG5-HESX1, resulting in a greater inhibition of PROP1-mediated gene stimulation.



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  		FIG. 2. Mutant HESX1 is enhanced in blocking PROP1 mediated gene transcription. A, A PRDQ9-luciferase construct (330 ng per well) was cotransfected into 293T cells in six-well plates with 60 ng/well of pSG5-PROP1, resulting in a 6- to 7-fold stimulation of luciferase activity (top bar). These constructs were cotransfected with 0–60 ng/well of wild-type (black bars) or mutant (hatched bars) pSG5-HESX1. Each well also received 7 ng of a Renilla luciferase vector to control for transfection efficiency. The data are expressed as relative luciferase activity (±SEM), with 1.0 defined as the luciferase activity in the presence of PRDQ9-luciferase alone, in the absence of cotransfected pSG5-PROP1 or pSG5-HESX1. B, Either 2 or 10 µg of pSG5-HESX1 or pSG5-mutant HESX1 was transfected into 293T cells. Proteins from cell extracts were obtained 48 h after transfection, and separated by SDS-PAGE. A Western blot analysis was performed using an anti-FLAG antibody. C, Either 2 or 10 µg of pSG5-HESX1 or pSG5-mutant HESX1 was transfected into 293T cells. mRNA was isolated and analyzed by Northern blot using an N-terminal HESX1 probe.

 
Cotransfection of pSG5-mutant HESX1 with pSG5-PROP1 also results in an inhibition of the PROP1 effects. Surprisingly, however, mutant HESX1 is stronger than wild-type HESX1 in this respect. For example, 0.5–1.0 ng of mutant HESX1 represses transcription to a similar level as 15 ng of the wild-type construct. In fact, mutant HESX1 is able to decrease the PROP1 effects back to baseline (i.e. luciferase activity in the absence of transfected PROP1), which is not even accomplished by high doses of wild-type HESX1. These data suggest that mutant HESX1 is more potent than the wild-type protein in blocking PROP1 activity.

A Western blot analysis using an anti-FLAG antibody was performed to assess the relative levels of wild-type and mutant HESX1. As shown in Fig. 2BGo, mutant HESX1 is not produced in greater amounts than wild-type HESX1. Either 2 or 10 µg of pSG5-wild-type or mutant HESX1 was transfected into cells; proteins from cell extracts were obtained, separated by SDS-PAGE, and analyzed by Western blot. In fact, there is actually a slight decrease in the amount of mutant protein produced. Therefore, the increased potency of the mutant protein is not due to increases in protein production. To assess whether the decreased amount of mutant HESX1is due to altered mRNA levels, either wild-type or mutant pSG5- HESX1 was again transfected into 293T cells, and mRNA was isolated. A Northern blot analysis using an amino-terminal portion of the HESX1 cDNA (in common to both the wild-type and mutant cDNA) was performed (Fig. 2CGo). Similar to the Western blot, the Northern blot reveals a decreased amount of mutant HESX1 mRNA. In sum, the increased mutant HESX1 effects are not explained by an increase in levels of protein (or mRNA) in the cells.

Mutant HESX1 binds DNA better than wild-type HESX1

To identify the mechanism of the increased potency of the mutant HESX1, EMSA was used to examine protein-DNA binding using a radiolabeled PRDQ9 probe. PROP1, HESX1, and mutant HESX1 were produced by a coupled in vitro transcription/translation reaction. As shown in Fig. 3AGo, increasing amounts of PROP1 result in a shift of radioactivity (lanes 1–3), indicating that PROP1 binds the PRDQ9 element, as expected. Similarly, HESX1 (lanes 4–6) and mutant HESX1 (lanes 7–9) also bind PRDQ9. However, the mutant HESX1 binds much better than the wild-type protein (compare lanes 5 and 8, or 6 and 9).



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  		FIG. 3. Mutant HESX1 binds a PRDQ9 element better than wild-type HESX1. A, HESX1, mutant HESX1, and PROP1 cDNA constructs in pSG5 were subjected to a coupled in vitro transcription/translation reaction in reticulocyte lysate using T7 polymerase. Two, 4, or 8 µl of proteins were incubated with a radiolabeled PRDQ9 probe for 20 min at room temperature, then run on a 5% nondenaturing gel. B, HESX1, mutant HESX1, and PROP1 cDNA constructs in pSG5 were subjected to a coupled in vitro transcription/translation reaction in reticulocyte lysate using T7 polymerase. Four microliters of unprogrammed reticulocyte lysate (UP), wild-type HESX1, mutant HESX1, or PROP1 were used in lanes 1–4, respectively. HESX1 (wild-type and mutant) and PROP1 were added together in lanes 5–6 to investigate heterodimerization; 2 µl of wild-type or mutant HESX1 (as indicated) and 2 µl PROP1 were added together and incubated with the PRDQ9 probe, to keep the total amount of protein constant. C, In vitro transcription/translation was performed in the presence of 35S-methionine with pSG5- wild-type and mutant HESX1. Samples were analyzed by SDS-PAGE and subjected to autoradiography. D, A UAS-Luciferase reporter (660 ng) was cotransfected with 150 ng of Gal4 (empty vector), Gal4-Hesx1, or Gal4-mutant Hesx1. Each well also received 30 ng of a CMV-ß-galactosidase expression vector to control for transfection efficiency. Data are expressed as relative luciferase activity (±SEM), where 1.0 is defined by the luciferase activity of Gal4 (empty vector). E, Transfection was performed with Gal4-wild-type Hesx1 and Gal4-mutant Hesx1 (as in D), but with the additional cotransfection of 660 ng pSG5 (empty vector) or pSG5-PROP1. Data are expressed as relative luciferase activity (±SEM), where 1.0 is defined as the luciferase activity of Gal4-wild-type Hesx1 with empty vector pSG5 (without PROP1). Transfection efficiency was controlled using a CMV-ß-galactosidase expression vector.

 
To determine whether the differences in DNA binding are also present when HESX1 heterodimerizes with PROP1, another EMSA was performed (Fig. 3BGo). Again, the mutant HESX1 binds much better than wild-type HESX1 (compare lanes 2 and 3). When PROP1 is added, both HESX1 proteins form heterodimers, which are intermediate in size between the HESX1 homodimers and the PROP1 homodimers (see lanes 5 and 6). This confirms that the mutation does not interfere with heterodimerization of HESX1 with PROP1. Furthermore, the mutant HESX1-PROP1 heterodimer binds DNA stronger than the wild-type HESX1-PROP1 heterodimer (lanes 5–6). To confirm that these differences in EMSA were not due to differences in protein generated, 35S-labeled wild-type and mutant HESX1 were analyzed by SDS-PAGE (Fig. 3CGo); in fact, no differences were found to explain the EMSA findings. Therefore, the increase in functional activity seen with the mutant HESX1 (Fig. 2AGo) is correlated with its increased ability to bind DNA (Fig. 3Go, A and B).

To determine whether the mutant HESX1 has greater intrinsic repression capabilities, both wild-type and mutant HESX1 were cloned down-stream of the Gal4-DNA binding domain. These constructs were transfected into 293T cells with a UAS-Luciferase reporter, which contains binding sites for Gal4, but not HESX1 or PROP1. In this way, the abilities of these proteins to repress gene transcription could be studied independently of their abilities to bind DNA. As shown in Fig. 3DGo, both wild-type and mutant HESX1 repress luciferase activity 3- to 4-fold, compared with empty vector Gal4. In particular, Gal4-mutant HESX1 is not able to repress transcription better than Gal4-wild-type HESX1, suggesting that the increased repression noted in Fig. 1Go is not due to greater interactions with corepressors. Finally, to examine interactions with PROP1, these constructs were additionally cotransfected with pSG5-PROP1. As shown in Fig. 3EGo, cotransfection with pSG5-PROP1 resulted in an increase in luciferase activity for both constructs, but was greater for Gal4-mutant Hesx1, suggesting that the mutant protein interacts better with PROP1. Therefore, mutant HESX1 blocks PROP-1-mediated transcriptional activation better than wild-type HESX1, not because of an increase in its intrinsic repression capabilities, but because it binds DNA better than the wild-type protein, and more avidly heterodimerizes with PROP1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although repressors are known to play vital roles in the development of many tissues, their role in pituitary development is not fully defined. HESX1 is the transcription factor most clearly identified as a repressor in pituitary organogenesis. HESX1 appears to be important in defining the timing of the initiation of the PROP1-dependent gene program (19). PROP1 is a related paired-like homeodomain transcription factor that functions as an activator and is important in the development of POU1F1 (Pit-1)-dependent cell lineages. It was therefore termed "Prophet of Pit-1," or PROP1 (20). There are two PROP1 binding sites in the -5-kb to -10-kb region of the POU1F1 flanking region; these were identified based on sequence homology to the PRDQ9 and related paired homeodomain factor binding elements (20). POU1F1 itself is necessary for the development of the somatotroph, lactotroph, and thyrotroph cell lineages, and mutations in both POU1F1 and PROP1 have been identified in a number of patients with combined pituitary hormone deficiency syndromes (2). POU1F1 binds to the coactivator cAMP response element binding protein-binding protein (CBP) (21, 27, 28) and transactivates the PRL, GH, and TSH genes. Interestingly, POU1F1 also binds the NCoR (21), although the physiological role of this interaction is not yet clear. However, this suggests that POU1F1 might act as a repressor under certain circumstances. In fact, one report suggests that the binding of NCoR to POU1F1 may allow repression of GH gene transcription in pituitary lactotrophs (29).

Hesx1 mediates repression by binding to TLE1 and NCoR (19, 21, 29). Through the actions of these corepressors, HESX1 appears to block PROP1-dependent gene transcription until HESX1 expression itself is attenuated (19). When there is little to no HESX1 present, PROP1 homodimers form, and the PROP1-dependent pituitary program can commence. Interestingly, if PROP1 is expressed too soon, the pituitary gland does not develop (19). Similarly, defective PROP1 function causes hypopituitarism in both mice and humans (10, 20). These data suggest that there must be careful timing of PROP1 action for normal pituitary development to occur. In the patient in the current studies, this timing would be expected to be abnormal (see below).

HESX1 may have additional roles besides its effects on PROP1 function. HESX1 mutations are associated with SOD, and the current patient had an absent corpus callosum and optic nerve hypoplasia. HESX1 appears to mediate forebrain development, and our data suggest that the HESX1 C terminus may play a role on this process, perhaps by interacting with a brain-specific coregulator. However, the identification of such factors is beyond the scope of this manuscript. In addition, HESX1 may also have effects on pituitary development beyond its modulation of PROP1 activity. For instance, a recent report suggests that HESX1 may repress transcription of the {alpha}-subunit and LHß promoters, with HESX1 binding the {alpha}-promoter as a monomer and LHß promoter as a dimer (30). The PRDQ9 element was chosen for this study, however, because clinically the patient manifested GH deficiency, which might be expected as a result of PROP1 dysfunction. Gonadotropin deficiency cannot be determined due to the patient’s prepubertal age. Interestingly, although there is no clear evidence of TSH deficiency in this patient, the thyroid hormone level was borderline when last checked. It is still too early, though, to determine whether this patient will develop central hypothyroidism.

Mutations in HESX1, which impair DNA binding, have been shown to result in variable degrees of hypopituitarism and SOD (23, 24, 25). The pituitary defects can range from isolated GH deficiency (23) to panhypopituitarism. Interestingly, targeted disruption of the mouse Hesx1 gene also results in a similar phenotype (19, 24). Heterozygous deletion of the mouse Hesx1 gene results in a mild phenotype in about 1% of animals (24). Presumably, in knockout mice (and in patients without HESX1), PROP1 is allowed to function prematurely, with deleterious consequences for pituitary development. Generally, heterozygous mutations in humans have resulted in more modest phenotypes than homozygous mutations (23), similar to the mouse models. Interestingly, all previous mutations of HESX1 identified have rendered mutant proteins with decreased activity. In contrast, we have identified a patient with SOD and GH deficiency with a mutation in HESX1 that enhances DNA binding and results in increased interactions with PROP1. This results in a mutant protein with more potent functional activity and an increased ability to block PROP1-dependent actions. This is the first mutation causing enhanced repression as an etiology of a congenital pituitary disorder. This is likely to be of most significance when HESX1 is normally being down-regulated. Under these circumstances, HESX1 would have minimal effects, but the mutant protein would still exhibit increased activity and would still be able to heterodimerize with PROP1. In this way, the effect of HESX1 would be extended, and normal PROP1 action would be delayed (Fig. 4Go). Thus, the timing of PROP1 action would be altered.



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  		FIG. 4. Model of mutant HESX1 effects on the PROP1-dependent program during pituitary development. Initially, HESX1 is expressed in the absence of PROP1. After PROP1 begins to be expressed, HESX1-PROP1 heterodimers are formed, which are inactive. As PROP1 expression increases and HESX1 expression decreases, PROP1 homodimers predominate, and PROP1-dependent gene transcription increases. The mutant HESX1 binds better to DNA response elements than the wild-type proteins. Therefore, even at low protein levels, mutant HESX1-PROP1 heterodimers are preferentially formed in place of PROP1 homodimers. This results in abnormal timing of the PROP1-dependent pituitary program.

 
In fact, transgenic mouse models have shown that prolonged expression of Hesx1 and TLE1 results in a severe pituitary phenotype, with the absence of all ventral pituitary cell types (19). In a transgenic model of prolonged expression of Hesx1 alone, though, there was a more mild phenotype, with a modest reduction of some cell lineages at E16.5 (19). In that study, pituitary-specific gene expression was not assessed postnatally, and GH expression specifically was not assessed. Interestingly, the patient in this study also has a relatively modest pituitary phenotype, as expected from the transgenic model in which mouse Hesx1 expression is prolonged but TLE1 expression is normal.

In the current study, the more potent repression caused by the mutant HESX1 initially could have been due to increased DNA binding or enhanced corepressor interactions. EMSA analysis reveals that the mutant HESX1 binds DNA better than the wild-type protein as both a HESX1 homodimer and a HESX1-PROP1 heterodimer. Interestingly, the HESX1-PROP1 heterodimer (both wild-type and mutant) appears to bind DNA better than either the HESX1 or PROP1 homodimers themselves. Furthermore, the transient transfection data suggest that even small amounts of HESX1 repress PROP1-mediated gene stimulation. These data suggest that the mutant HESX1 results in more stable protein-DNA complexes, and may have an increased ability to titrate PROP1 away from the formation of (active) PROP1 homodimers (Fig. 4Go). This would delay the initiation of the PROP1 effects on POU1F1 and potentially other pituitary-specific promoters. In fact, the increased binding itself would be expected to result in increased corepressor recruitment to the DNA promoter regions, even in the absence of intrinsic differences in corepressor interactions. Interestingly, the mutation of HESX1 identified in the current study is C-terminal to the HESX1 homeodomain. Because TLE1 and NCoR bind HESX1 in the N terminus (19) and homeodomain (21), respectively, it is unlikely that the mutation will directly affect HESX1-corepressor interactions in the absence of alterations in HESX1 binding to its response elements. In fact, Gal4-mutant HESX1 was not able to repress gene transcription better than Gal4-wild-type HESX1 on a heterologous reporter, suggesting that differences in corepressor recruitment do not explain the functional effects. An underlying HESX1-PROP1 response element is therefore required.

In summary, we have identified a novel HESX1 mutation associated with GH deficiency and SOD that results in increased DNA binding and enhanced repression of gene activity. These data support a model in which enhanced HESX1 activity results in a delay in PROP1 action leading to pituitary abnormalities. These data are also in agreement with previous in vivo transgenic mouse models suggesting that the action of HESX1 in the developing pituitary must be carefully timed. Finally, enhanced activity by transcriptional repressors should be considered a potential mechanism for pituitary and other developmental abnormalities.


   Acknowledgments
 
We thank Fredric Wondisford for discussions.


   Footnotes
 
This work was supported by grants from the National Institutes of Health (to R.N.C., L.E.C., and S.R.) and the Genentech Foundation for Growth and Development (to L.E.C.). M.J. was the recipient of a Howard Hughes Medical Institute Summer Student Research Fellowship.

Abbreviations: CMV, Cytomegalovirus; E13, embryonic d 13; NCoR, nuclear corepressor; PRL, prolactin; SOD, septooptic dysplasia; UAS, upstream activating sequence.

Received November 27, 2002.

Accepted July 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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